Invariant natural killer T (iNKT) cells are innate-like T lymphocytes cells that recognize glycolipid antigens associated with CD1d, non-classical antigen presenting proteins. They can drive either pro-inflammatory (Th-1) or anti-inflammatory (Th-2) immune microenvironment through the production of both Th-1 and Th-2 type cytokines upon activation, thus play a vital role in cancer, infection, and autoimmune diseases. Adoptive cell therapy using ex vivo expanded iNKT cells is a promising approach to enhance anti-tumor immunity or immunosuppression. However, overcoming phenotypic and functional heterogeneity and promoting in vivo persistency of iNKT cells remains to be a challenge. Here, we compared various methods for ex vivo expansion of human iNKT cells and assessed the quality of expansion, phenotype, and cytokine production profile of expanded iNKT cells. While a direct stimulation of iNKT cells in peripheral blood mononuclear cells with agonist glycolipid led to the expansion of iNKT cells in varying degrees, stimulation of enriched iNKT cells by irradiated autologous peripheral blood mononuclear cells or allogeneic dendritic cells resulted in consistent expansion of highly pure iNKT cells. Interestingly, the mode of antigenic stimulation influenced the dominant subtype of expanded iNKT cells. Further, we evaluated whether additional IL-7 or IL-15 during antigenic stimulation with allogeneic dendritic cells can improve the phenotypic heterogeneity and modify cytokine production profile of iNKT cells expanded from 18 consecutive donors. The presence of IL-7 or IL-15 during antigenic stimulation did not affect the fold of expansion or purity of expanded iNKT cells. However, IL-7, but not IL-15, led to a better expansion of CD4⁺ iNKT cells, enhanced Th-2 type cytokine production of CD4⁺ iNKT cells, and maintained the expansion of central memory (CD45RA^-CD62L⁺) CD4⁺ iNKT cells. Our results suggest the addition of IL-7 during antigenic stimulation with allogeneic dendritic cells can promote the expansion of CD62L⁺Th-2⁺CD4⁺ human iNKT cells that can be used as novel immunotherapeutic to control excessive inflammation to treat various autoimmune diseases.

不变的自然杀伤性T细胞（iNKT）是先天性T淋巴细胞，可识别与CD1d，非经典抗原呈递蛋白相关的糖脂抗原。它们可以在激活后通过产生Th-1和Th-2型细胞因子来驱动促炎（Th-1）或抗炎（Th-2）免疫微环境，因此在癌症，感染，和自身免疫性疾病。过继细胞疗法离体扩增的iNKT细胞是增强抗肿瘤免疫或免疫抑制的一种有前途的方法。但是，克服表型和功能异质性并促进体内iNKT细胞的持久性仍然是一个挑战。在这里，我们比较了各种方法离体iNKT细胞的扩增，并评估了扩增的iNKT细胞的扩增质量，表型和细胞因子的产生。尽管激动剂糖脂直接刺激外周血单核细胞中的iNKT细胞导致iNKT细胞不同程度的扩增，但辐照的自体外周血单核细胞或同种异体树突状细胞刺激富集的iNKT细胞却导致了高纯度iNKT的持续扩增。细胞。有趣的是，抗原刺激的模式影响了扩展的iNKT细胞的主要亚型。此外，我们评估了同种异体树突状细胞在抗原刺激过程中是否需要额外的IL-7或IL-15可以改善表型异质性并改变从18个连续供体中扩增的iNKT细胞的细胞因子产生情况。抗原刺激过程中IL-7或IL-15的存在不会影响iNKT细胞扩增的倍数或纯度。但是，IL-7而非IL-15导致CD4更好的扩展⁺ iNKT细胞，增强的Th-2型细胞因子的产生的CD4 ⁺ iNKT细胞，并保持中枢记忆的膨胀（CD45RA ^- CD62L ⁺）CD4 ⁺ iNKT细胞。我们的结果表明，在用同种异体树突状细胞进行抗原刺激过程中添加IL-7可以促进CD62L ⁺ Th-2 ⁺ CD4 ⁺人iNKT细胞的扩增，这些细胞可以用作新型免疫疗法来控制过度炎症以治疗各种自身免疫性疾病。